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PubMed2006

Trichinella nativa and Trichinella T9 in the Hokkaido island, Japan.

Trichinella sp. muscle larvae were isolated from the thigh muscle of two red foxes (Vulpes vulpes) captured in Sapporo and Otofuke, Hokkaido, Japan, in 2003. Multiplex PCR designed for genotyping the genus Trichinella revealed that the Sapporo isolate showed a specific pattern to T. britovi complex (T. britovi, Trichinella T8 and Trichinella T9) and the Otofuke isolate showed that to T. nativa. Nucleotide sequences of a part of the mitochondrial cytochrome oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) of the Sapporo isolate showed the highest similarity to those of Trichinella T9, a species detected in the mainland of Japan. This study shows that both T. nativa and Trichinella T9 are circulating in wildlife of the Hokkaido island.

Animals↗

PubMed2005

[Enterococcal bacteremia at Nîmes university hospital].

PURPOSE OF THE STUDY: The aim of this study was the description of enterococcal bacteremia and the evaluation of their resistance evolution to antibiotics and the virulence profile of these isolates. PATIENTS AND METHODS: We have studied all the bacteremia caused by Enterococcus spp. from the 1st of July 2003 until the 30th of June 2004 at Nîmes university hospital. For each isolate, population, clinical and microbiological data were collected. Multiplex PCR allowed for the identification of glycoprotein resistant phenotypes and furthermore, to identify the main virulence genes found in the enterococci. RESULTS: Thirty-three strains were identified from 33 patients (60.6% male) with a median age of 68 years (median: 3-96). The vital prognostic was calculated in 63.6% of the cases (MacCabe > or =1); seven patients died during their hospitalization (21.2%). The infections were nosocomial in 60.6% of the cases. No epidemic was observed. The principal comorbidities were cardiovascular and digestive pathologies. In addition, 54.5% of patients had had a surgical intervention. Enterococcus faecalis (87.9%) was the cause of infection in the majority of cases followed by Enterococcus faecium. Most of the infections were monomicrobial (75.8%). E. faecalis was sensitive to amoxicillin and to glycopeptides but was resistant to norfloxacin, erythromycin and pristinamycin. The same observations were made for the E. faecium with the exception of amoxicillin. E. faecalis had the maximum number of virulence genes and E. faecium the least. CONCLUSION: The enterococcal bacteremia had mainly a nosocomial origin. The isolates came from digestive tract flora but also from catheters. The mortality rate was 21.2%. A developing sensitivity to erythromycin and pristinamycin was observed in comparison to the data of the ONERBA (2002-2003). Only one isolate was resistant to glycopeptide. This was the first European E. faecium containing the vanD allele. This isolate proved fatal to the patient. The dark evolution of enterococcal bacteremia and their frequent nosocomial origin induce to promote studies intended for better knowing the risk factors of such infections.

Adolescent↗

PubMed2005

Detection of Salmonella in environmental water and sediment by a nested-multiplex polymerase chain reaction assay.

From 1995 to 2002, 53 serovars of Salmonella were isolated in the Seine estuary (France). The 3 serovars most frequently found were S. enterica serovar Typhimurium, S. enterica serovar Infantis and S. enterica serovar Virchow. A nested multiplex PCR (nm-PCR) assay was developed to detect the presence of Salmonella in estuarine water and sediment samples. The target gene used was the phase 1 flagellin fliC chromosomal gene, present in all Salmonella serovars. A set of 4 primers was first used to amplify an 890-bp sequence of the fliC gene, and then a second set of 3 primers was used for the nested PCR. The nmPCR method has been successfully tested for 28 serovars, 13 of which are of epidemiological significance. The detection limit of the assay, without any pre-enrichment step, was estimated at 1 CFU in deionized water, and at 4-5 CFU in the reaction mixture when tested on estuarine water seeded with a Salmonella strain. When the nmPCR was used together with the classical culture method in environmental samples, it gave additional positive results for 11.3% of the sediment samples and 20% of the water samples despite a high background of other bacteria. Overall, the results demonstrated that this molecular approach informed us about the contamination by Salmonella of estuarine water and sediment samples. Positive amplifications suggested the presence of Salmonella DNA and could thus provide information about a recent (culturable) or past (non-culturable, released DNA) contamination of environmental samples by this pathogenic bacteria.

DNA Primers↗

PubMed2026

Targeted variant analysis of feline mediastinal lymphoma using MassARRAY and clinical associations.

Lymphoma is the most commonly diagnosed cancer in cats. This study used the Agena MassARRAY to genotype 40 variants across 17 genes in feline mediastinal lymphoma. These variants have previously been identified in tumors, including T- and B-cell lymphomas, acute and chronic lymphocytic leukemias, and mast cell tumors, in humans, dogs, and cats, using various methods. They were selected based on high prevalence reported in prior oncology studies, potential relevance to targeted therapy, and suitability for multiplex PCR amplification. Pleural fluid samples were collected from 76 cats with mediastinal lymphoma, including 69 domestic shorthairs, two Persians, two Siamese, two Wichienmaat, and one Scottish Fold. The most prevalent variants were found in the BCL2, KIT, STAT3, and ZEB1 genes. Specifically, BCL2 c.83275986G > A and c.83275992G > T were present in 71.1% and 57.9%, respectively. In cats with variant-positive in KIT c.163965724C > CT significantly reduced (11 days) compared to wild-type cats (94 days) (p < 0.001). In cats with variant-positive in STAT3 c.42942437C > CA, resulted in shorter median survival compared to wild-type cats (18 days vs. 77 days, p = 0.006). The findings suggest that the variant panel could be useful for the genomic landscape of feline mediastinal lymphoma and warrant further validation.

Animals↗

PubMed2004

Polymorphisms of cytochrome P450 1A1, glutathione S-transferases M1 and T1 in a Turkish population.

Intra-ethnic as well as inter-ethnic differences are known to exist in the frequencies of cytochrome P450 (CYP) 1A1, glutathione S-transferase (GST) M1, and GSTT1 gene polymorphisms with which associations have been shown for several cancers. In this study, CYP1A1 m2, GSTM1, and GSTT1 gene polymorphisms were determined among 133 healthy individuals of a Turkish population. On the basis of polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) methodology, the frequency of CYP1A1 m2 mutation was determined. The multiplex PCR protocol was used to determine the frequency of the deleted genotypes of both GSTM1 and GSTT1 genes. The frequencies of Ile/Ile (wild type), Ile/Val (heterozygous variant), and Val/Val (homozygous variant) CYP1A1 m2 genotypes were 90.2%, 9.8%, and 0%, respectively. The frequencies of the deleted GSTM1 (null) and GSTT1 (null) genotypes were 51.9% and 17.3%, respectively. These results show that the frequencies of the CYP1A1 m2, GSTM1, and GSTT1 gene polymorphisms in a Turkish population are similar to Caucasian populations.

Adult↗

PubMed2006

Identification of two subpopulations of Bacillus Calmette-Guérin (BCG) Tokyo172 substrain with different RD16 regions.

Two types of colonies with different morphologies (smooth: S and rough: R) formed when Bacillus Calmette-Guérin (BCG) Tokyo172 substrain was cultured on Middlebrook 7H10 agar medium, and their genotypes were analyzed by multiplex PCR on five RD regions and SenX3-RegX3. In most cases these two colony types had different genotypes, i.e., S colonies showed a characteristic 22 bp deletion in Rv3405c of the RD16 region (type I), and R colonies did not have this deletion (type II) similar to many other BCG substrains. Thus, there was a strong relationship between colony morphology and genotype. Both genotypes were found in every Tokyo172 preparation tested, including the seed lot for production, the origin of seed lot from the 1960s and ATCC BCG Japan. Type I was always in the majority. It was suggested that types I and II constituted independent subpopulations within the Tokyo172 substrain. Type I was shown to have a growth advantage over type II both on culture media and in mice organs.

Animals↗

PubMed2004

Evaluation of the German cockroach (Blattella germanica) as a vector for verotoxigenic Escherichia coli F18 in confined swine production.

German cockroaches are common pests of confined swine production in North Carolina and other southeastern states. Vector competence of German cockroaches for one of the most important porcine bacterial pathogens, verotoxigenic Escherichia coli F18, was evaluated in laboratory bioassays using a culturing approach followed by multiplex PCR. In addition, the populations of fecal coliforms from the feces of piglets and cockroaches collected from a swine nursery were assessed. Viable and virulent cells of E. coli F18 were detected in cockroach feces for up to 8 days after the initial exposure. The population of fecal coliforms in cockroach feces was high (4.4 x 10(5) CFU g(-1)) and comparable to that of piglet feces (1.9 x 10(6) CFU g(-1)). This study demonstrates that cockroaches may serve as important mechanical vectors of pathogenic E. coli. Integrated management of cockroach populations should be incorporated into the disease prevention and control programs in the swine industry.

Animals↗

PubMed2005

Exudative epidermitis in pigs caused by toxigenic Staphylococcus chromogenes.

Staphylococcus chromogenes is closely related to Staphylococcus hyicus, which is recognised as the causative agent of exudative epidermitis (EE) in pigs. S. chromogenes is part of the normal skin flora of pigs, cattle and poultry and has so far been considered non-pathogenic to pigs. A strain of S. chromogenes producing exfoliative toxin type B, ExhB, was identified by the use of a multiplex PCR specific for the exfoliative toxins from S. hyicus. The exfoliative toxin from S. chromogenes reacted in immunoblot analysis with polyclonal and monoclonal antibodies specific to ExhB from S. hyicus and had an apparent molecular weight of 30 kDa. Sequencing the gene encoding the exfoliative toxin from S. chromogenes revealed that the molecular weight of the toxin with the signal peptide and the mature toxin was 30,553 and 26,694 Da, respectively. Comparison of the exhB genes from S. chromogenes strain VA654 and S. hyicus strain 1289D-88 showed differences in seven base pairs of the DNA sequences and in two amino acid residues in the deduced amino acid sequences. Pigs were experimentally inoculated with S. chromogenes strain VA654. By clinical observations and histopathological evaluation of the skin alterations, all pigs revealed development of generalized exudative epidermitis. No toxin producing S. hyicus was isolated from the pigs and all ExhB-positive bacterial isolates were identified as S. chromogenes. This confirmed that the disease-causing agent was the inoculated S. chromogenes strain VA654. The results of this study show that S. chromogenes may cause exudative epidermitis in pigs.

Amino Acid Sequence↗

PubMed2004

Characterisation of Trichinella isolates from Bulgaria by molecular typing and cross-breeding.

Trichinella spp. larvae were collected from domestic and wild-life animals in association with 15 human trichinellosis outbreaks registered between 1999-2002 in Bulgaria. Furthermore, Trichinella spp. isolates were obtained from 62 naturally infected wild animals and of a rat. All isolates were subjected to speciation by both multiplex PCR and cross-breeding experiments. Epidemiological and clinical data were collected and analysed using standard protocols for epidemiological surveillance and control of outbreaks. Only two species were identified-Trichinella britovi and Trichinella spiralis. Results obtained by molecular typing fully matched those of cross-breeding. More specifically, parasite isolates obtained upon 15 epidemic outbreaks revealed the predominance of T. britovi (n = 10) when compared to T. spiralis (n = 5). With regard to host origin, the predominant species detected among wild boar was T. britovi (n = 4), and T. spiralis was identified in one wild boar sample only. Among the isolates obtained from domestic pig products, T. britovi was found in five cases and T. spiralis in four cases, respectively. In the naturally infected wild animals not related to epidemics, only T. britovi was demonstrated. The present results provide a strong indication that both T. britovi and T. spiralis operate within domestic and sylvatic cycles in Bulgaria. Geographically, the distribution of T. britovi appears to include Central, Southern, Eastern and Western parts of the country, and wildlife animals from the Mid Balkan Mountains and Mid Sredna Gora Mountains, T. spiralis was found in Western and Southwestern Bulgaria, only.

Animals↗

PubMed2004

Inappropriate feeding practice favors the transmission of Trichinella papuae from wild pigs to saltwater crocodiles in Papua New Guinea.

The recent discovery of Trichinella zimbabwensis in farmed crocodiles (Crocodilus niloticus) of Zimbabwe and its ability to infect mammals, and the development of both T. zimbabwensis and Trichinella papuae in experimentally infected reptiles led to an investigation of Trichinella infection in saltwater crocodiles (Crocodylus porosus) and in wild pigs (Sus scrofa) of Papua New Guinea, to see if T. papuae also, is present in both cold- and warm-blooded animals. Of 222 crocodiles examined, 47 animals (21.2%), all from Kikori, Gulf Province, were positive for non-encapsulated larvae in the muscles. The greatest number of larvae was found usually in the biceps, with an average of 7 larvae/g. One isolate from a crocodile infected successfully both laboratory rats and mice. Of 81 wild pigs examined, 9 from Bensbach river area (Western Province) and 1 from Kikori area (Gulf Province) were positive for non-encapsulated larvae in the muscles. Trichinella larvae from both saltwater crocodiles and wild pigs have been identified by multiplex-PCR analysis as T. papuae. The sequence analysis of the region within the large subunit ribosomal DNA, known as the expansion segment V, has shown the presence of a molecular marker distinguishing T. papuae isolates of Bensbach river area from those of Kikori area. This marker could be useful to trace back the geographical origin of the infected animal. The epidemiological investigation carried out in the Kikori area has shown that local people catch young crocodiles in the wild and keep them in holding pens for several months, before sending them to the crocodile farm in Lae (Morobe Province). They feed the crocodiles primarily with wild pig meat bought at the local market and also with fish. These results stress the importance of using artificial digestion for routinely screening of swine and crocodiles, and of adopting measures for preventing the spread of infection, such as the proper disposal of carcasses and the adequate freezing of meat.

Alligators and Crocodiles↗

PubMed2005

Improved methods for isolating DNA from Ostertagia ostertagi eggs in cattle feces.

A multiplex PCR assay for differentiating strongyle eggs from cattle has recently been described; however, the egg disruption and DNA extraction procedures, though effective, are inadequate for large studies or clinical application. The purpose of this research was to evaluate methods for disrupting trichostrongyle eggs, then assess commercial kits for extracting egg DNA using Ostertagia ostertagi as a model species. Egg disruption procedures tested included probe sonication, bath sonication, bead beating, boiling, microwaving, proteinase K/SDS digestion, freezing, and various combinations of the above with the incorporation of sodium dodecyl sulfate. These procedures were evaluated in conjunction with four commercial DNA extraction kits: DNA Stool mini kit and DNeasy Plant kit (Qiagen), Fast DNA kit (QBiogene), and the MAP extraction kit (Tetracore). Results showed that egg disruption was best accomplished with the bead beater and ceramic beads, resulting in 100% disruption within 1min. When DNA extraction was preceded by the isolation of eggs from feces, all procedures except the Fast DNA kit produced PCR-ready DNA from at least two eggs. The DNeasy Plant kit allowed consistent detection of DNA released from one egg. Due to the morphological similarities among trichostrongyle eggs in ruminants, strongyle eggs in equids, and hookworm eggs, the methods described herein may have broad application to other nematodes.

Animals↗

PubMed2006

A Trichinella murrelli infection in a domestic dog in the United States.

Trichinella murrelli infection was diagnosed in a naturally infected Beagle bitch from VA, USA, where encapsulated larvae were found in histological sections of several skeletal muscles. A laboratory reared dog fed infected muscles resulted in viable muscle larvae that were subsequently infective to Swiss-Webster mice. Multiplex PCR using larvae from the experimentally infected dog demonstrated two distinct bands migrating at 127 bp and 316 bp which together are diagnostic for T. murrelli; the isolate was assigned the ISS code: ISS1608 by the International Trichinella Reference Centre. This is the first report of T. murrelli infection in a companion animal.

Animals↗

PubMed2006

Trichinella spiralis and Trichinella pseudospiralis mixed infection in a wild boar (Sus scrofa) of Germany.

A wild boar (Sus scrofa) from the island Usedom in Mecklenburg-Western Pomerania (north-east Germany) was detected as Trichinella-positive during routine meat inspection. Encapsulated and non-encapsulated larvae were detected in the muscle tissue by trichinoscopy. In the diaphragm, 922 larvae per g were detected by artificial digestion. Muscle larvae displayed two different sizes of about 700 and 1100 microm. By a multiplex PCR analysis, larvae with a large size were identified as Trichinella spiralis, whereas those of a smaller size were identified as Trichinella pseudospiralis. This is the first finding of a mixed infection of T. spiralis and T. pseudospiralis in a naturally infected animal and it supports the tendency of more frequent detection of the non-encapsulated species T. pseudospiralis in Europe.

Abattoirs↗

PubMed2006

Epidemiological investigations on Trichinella infections in farmed fur animals of Estonia.

Farming of fur animals represents an important income in Estonia. Even though Trichinella worms does not induce a symptomatic disease in carnivores, the carcasses of skinned animals can increase the biomass of the parasite in the environment, if they are not properly destroyed. The aim of the present survey was to study the prevalence of Trichinella worms in farmed fur animals of Estonia. Of 281 muscle samples from blue foxes (Alopex lagopus), silver foxes (Vulpes vulpes fulva), minks (Mustela vison) and raccoon dogs (Nyctereutes procyonoides), which were collected in eight farms, Trichinella larvae have been detected in 21 animals (8%) from two farms by HCl-pepsin digestion. The highest number of larvae per gram of muscle was found in the front limb muscles. Larvae of the 21 isolates have been identified as Trichinella britovi or Trichinella nativa by a multiplex-PCR analysis.

Animals↗

PubMed2005

Prevalence of vaccine-derived polioviruses in sewage and river water in South Africa.

Polioviruses (PVs) are not associated with waterborne transmission to the same extent as many other enteric viruses. However, they are typically transmitted by the faecal-oral route, which implies that the risk of infection by exposure to the viruses in water cannot be underestimated. The risk appears particularly high for rural communities, which use sewage-polluted river water for domestic purposes. Thus, the presence in the environment of highly evolved, neurovirulent vaccine-derived poliovirus (VDPV) strains in the absence of polio cases would have important implications for strategies to terminate immunisation with oral poliovirus vaccine (OPV) following global polio eradication. The aim of the current study was to determine the prevalence of VDPVs in selected sewage and river water samples collected from 2001 to 2003, and to construct phylogenetic trees of the partially sequenced 5'untranslated region (5'UTR) and the VP1 region of the genomes to deduce the genetic relatedness between the PV strains. Using the monolayer plaque assay, 703 plaques from sewage and 157 plaques from river water samples were analysed. Application of a RT-multiplex PCR revealed that 176 of these plaques were non-polio enteroviruses, and 49 were PV isolates. The Sabin-specific RT-triplex PCR revealed the presence of 29 Sabin PV type 1, 8 Sabin PV type 2 and 12 Sabin PV type 3 isolates. The 5'UTR and the VP1 region of 13 PV type 1, 7 PV type 3 and 6 PV type 2 isolates were partially sequenced. The majority of the OPV isolates (24 out of 26) displayed close sequence relationships (>99% VP1 sequence identity) to the parental Sabin PV vaccine strains and were classified as "OPV-like viruses". Two isolates (D1 08/28 and OF1 05/21) were found to be highly divergent and were classified as "suspected" VDPVs. Isolate OF1 05/21 (a "suspected" VDPV type 1) showed more than 0.9% divergence in VP1, whereas isolate D1 08/28 (a "suspected" VDPV type 2) showed 1.4% divergence in VP1 from the parental Sabin PV vaccine strains. As with most of the other OPV-like isolates, these "suspected" VDPVs were carrying mutations, which have previously been associated with reversion of the attenuated Sabin PV strains to increased neurovirulence. It was estimated that the total period of replication for the two "suspected" VDPVs was between 12 and 16 months. In conclusion, this study provided new and relevant information on the prevalence of "suspected" VDPVs in sewage and river water, and opened the way to assess the possible broader significance of the findings reported here.

Base Sequence↗

PubMed2004

Genetic polymorphisms of GSTM1, p21, p53 and HPV infection with cervical cancer in Korean women.

OBJECTIVES: The aim of this study was to determine whether GSTM1 or GSTT1 might be associated with risk of cervical cancer development in Korean women. The multiplicative interaction of GSTM1 and GSTT1 genotype with p21, p53 polymorphism, and HPV genotype was also investigated. METHODS: From 1997 to 1999, uterine cervical carcinoma was diagnosed in 215 Korean women at the Department of Obstetrics and Gynecology of Seoul National University Hospital. None of the women in the control groups (n = 98) had any evidence of cervical lesions, which were confirmed by Pap smear. Finally, 81 cases and 86 controls were genotyped for p21, p53, and GSTM1 and T1 and HPV infection. A multiplex PCR method was used for the genotyping of GSTM1 and GSTT1; direct sequencing for p53 codon 72, high-risk HPV, and PCR-RFLP (BsmAI) for p21 codon. The unconditional logistic regression analysis was used to calculate ORs and 95% CI. RESULTS: Although the GSTM1 and GSTT1 genotype was not significantly associated with cervical cancer development for all women, the GSTM1 null genotype was significantly associated with an increased risk of cervical cancer development in women with high-risk HPV infection (OR = 2.9, 95% CI: 1.0-8.2). Although the frequency of overall GSTT1 null genotype was significantly lower in cervical carcinoma patients with high-risk HPV infection (OR = 0.3, 95% CI: 0.1-1.0), almost 2-fold increased risk was observed among women with GSTT1 null and Arg/Arg genotype (OR = 1.9, 95% CI: 0.7-5.4). Although the cervical cancer risk was 3.3-fold increased in women with null and Arg/Arg genotype compared to women with GSTM1 present and p21 Ser-containing genotype, there was no significant multiplicative interaction between GSTM1 and p21 (P for interaction = 0.785) or p53 (P for interaction = 0.815). CONCLUSIONS: These findings suggest that the risk of cervical cancer may be related to GSTM1 genotype in women with high-risk HPV infection and that there is a possible gene-gene interaction in the incidence of cervical cancer.

Female↗

PubMed2005

Glutathione S-transferase polymorphisms and ovarian cancer treatment and survival.

OBJECTIVE: Members of the glutathione S-transferase (GST) family have been shown to have functional polymorphisms that may affect drug metabolism and influence the effects of chemotherapy and survival from cancer. GSTM1, GSTT1, and GSTP1 genotypes were evaluated for their role in ovarian cancer treatment and survival. METHODS: DNA was extracted from tumor tissues of 215 patients diagnosed with primary epithelial ovarian cancer. GSTM1 and GSTT1 genotypes were determined by multiplex PCR; GSTP1 genotypes were assessed with PCR-RFLP. Associations between GST polymorphisms and risk of ovarian cancer progression or death were analyzed using Cox proportional hazards regression; subgroups of patients receiving different chemotherapeutics were also evaluated. RESULTS: GST polymorphisms were not found to be associated with patient or tumor characteristics or response to treatment. However, GSTM1 null patients were less likely to have disease progression (HR: 0.65, 95% CI: 0.43-0.99) or to die (HR: 0.68, 95% CI: 0.45-1.03) compared to patients with GSTM1. Patients with GSTM1 null and GSTP1 ile/val or val/val (reduced function) had a further reduction in risk of disease progression compared to patients with GSTM1 or GSTP1 ile/ile (HR: 0.42, 95% CI: 0.24-0.75). A similar association was also suggested for overall survival (HR: 0.61, 95% CI: 0.36-1.05). Subgroup analyses indicated that the effects of GST on survival were more pronounced among patients treated with specific chemotherapeutics. CONCLUSION: These findings support the idea that reduced GST function may improve ovarian cancer survival after post-operative chemotherapy; evaluation of GST functional polymorphisms may help to predict ovarian cancer prognosis.

Adult↗

PubMed2003

A simple and rapid method to determine the zygosity of uPA-transgenic SCID mice.

Successful transplantation of xenogeneic hepatocytes into uPA-transgenic SCID mice depends on the zygosity of the recipient mice. Normally, the difference between homozygous and heterozygous animals is determined via a quantitative Southern blot. We sequenced a part of the mouse genome that is eliminated upon integration of the transgene in the genome. Based on that sequence we developed a multiplex PCR that allows the unambiguous discrimination of negative, heterozygous, and homozygous uPA-transgenic SCID mice in a single day procedure. The speed of the procedure is an essential quality because transplantation of xenogeneic hepatocytes into uPA-SCID mice should be done as soon as possible after birth.

Animals↗